Mutation analysis of membrane type-1 matrix metalloproteinase (MT1-MMP) - The role of the cytoplasmic tail Cys(574), the active site Glu(240), and furin cleavage motifs in oligomerization, processing, and self-proteolysis ofMT1-MMP expressed in breast carcinoma cells

Membrane type-1 matrix metalloproteinase (MT1-MMP) is a key enzyme in the a ctivation pathway of matrix prometalloproteinase-2 (pro-MMP-8). Both activa tion and autocatalytic maturation of pro-MMP-2 in trans suggest that MT1-MM P should exist as oligomers on the cell surface. To better understand the f unctions of MT1-MIMP, we designed mutants with substitutions in the active site (E240A), the cytoplasmic tail (C574A), and the RRXR furin cleavage mot ifs (R89A, ARAA, and R89A/ ARAA) of the enzyme. The mutants were expressed in MCF7 breast carcinoma cells that are deficient in both MMP-2 and MT1-MMP , Our results supported the existence of MT1-MMP oligomers and demonstrated that a disulfide bridge involving the Cys(574) Of the enzyme's cytoplasmic tail covalently links MT1-MMP monomers on the MCF7 cell surface, The prese nce of MT1-MMP oligomers also was shown for the enzyme naturally expressed in HT1080 fibrosarcoma cells. The single (R89A and ARAA) and double (RS9A/A RAA) furin cleavage site mutants of MT1-MMP were processed in MCF7 cells in to the mature proteinase capable of activating pro-MMP-2 and stimulating ce ll locomotion. This suggested that furin cleavage is not a prerequisite for the conversion of pro-MT1-MMP into the functionally active enzyme. A hydro xamate class inhibitor (GM6001, or Ilomastat) blocked activation of MT1-MIM P in MCF7 cells but not in HT1080 cells. This implied that a matrixin-like proteinase sensitive to hydroxamates could be involved in a furin-independe nt, alternative pathway of MT1-MMP activation in breast carcinoma cells. Th e expression of the wild type MT1-MMP enhanced cell invasion and migration, indicating a direct involvement of this enzyme in cell locomotion. In cont rast, both the C574A and E240A mutations render MT1-MMP inefficient in stim ulating cell migration and invasion, In addition, the C574A mutation negati vely affected cell adhesion, thereby indicating critical interactions invol ving the cytosolic part of MT1-MMP and the intracellular milieu.